Biomarker for osteoarthritis and/or other ageing-related diseases, and use thereof

ABSTRACT

The invention relates to the identification of a biomarker whose abundance in biological samples is changed in subjects with osteoarthritis and/or other ageing-related diseases. The biomarker has applications in the diagnosis of osteoarthritis and/or other ageing-related diseases, in determining the prognosis for an individual diagnosed with osteoarthritis and/or other ageing-related diseases, and in monitoring the efficacy of treatment for osteoarthritis and/or other ageing-related diseases.

The present invention relates to a biomarker for osteoarthritis and/orother ageing-related diseases. In particular, the invention relates to amethod of diagnosing osteoarthritis, and/or other ageing-relateddiseases, by determining the level of a biomarker in a biologicalsample. The invention also relates to the use of a biomarker found inbiological samples to monitor the efficacy of a treatment forosteoarthritis, and/or other ageing-related diseases, and to determinethe prognosis for an individual diagnosed with osteoarthritis, and/oranother ageing-related disease.

Osteoarthritis is a progressive disorder characterized by destruction ofarticular cartilage and subchondral bone, and by synovial changes.Currently the diagnosis of osteoarthritis is based on clinical andradiographic changes which occur late during disease progression. Morespecifically, diagnosis is based on cartilage integrity, which asarticular cartilage is invisible on radiographs must be assessedindirectly from the spacing between subchondral bone ends in a joint.This method does not allow detection of early structural damage, and iscumbersome to use in daily practice.

Biochemical markers of bone, synovium or cartilage turnover have beenproposed as potential tools for the diagnosis, prognosis and treatmentmonitoring of osteoarthritis (Garnero, P., et al., Ann Rheum Dis, 2001.60 (6): p. 619-26; Bruyere, O., et al., J Rheumatol, 2003. 30 (5): p.1043-50; and Wu, J., et al., Arthritis Rheum, 2007. 56 (11): p.3675-84). More specifically, Wu, J., et al. (Arthritis Rheum, 2007. 56(11): p. 3675-84) describe potential molecular mediators and biomarkersof osteoarthritis in cartilage tissue. The method used requiredarticular cartilage to be obtained, which requires an invasive procedureand provides only a limited amount of tissue. The method is thereforecostly, time consuming and unsuitable for routine diagnostic testing, orfor monitoring disease progression, or for determining the therapeuticeffect of a treatment.

There therefore remains a need for a simple, rapid and effective methodfor the diagnosis of osteoarthritis and/or other ageing-relateddiseases, and/or to monitor the efficacy of treatments forosteoarthritis and/or other ageing-related diseases, and/or to determinethe prognosis for a patient diagnosed with osteoarthritis and/or otherageing-related diseases.

The present invention provides a method for (i) diagnosingosteoarthritis and/or another ageing-related disease, (ii) determiningthe prognosis for a patient with osteoarthritis and/or anotherageing-related disease, and (iii) monitoring the efficacy of treatmentfor osteoarthritis and/or another ageing-related disease, using readilyavailables which are simple to obtain and allow for rapid and costeffective use.

Reference herein to ageing-related diseases includes osteoporosis, andother degenerative diseases.

According to one aspect, the present invention provides a method ofdetermining the osteoarthritis status of a subject, and/or the status ofanother ageing-related disease in a subject, comprising the steps of:

-   -   (i) determining the concentration in a biological sample of a        peptide comprising the same or substantially the same amino acid        sequence as Seq ID no:1 or a fragment thereof;    -   (ii) comparing the peptide concentration determined in step (i)        with one or more reference values.

According to another aspect, the present invention provides a method ofdiagnosing osteoarthritis and/or another ageing-related disease in asubject, comprising the steps of:

-   -   (i) determining the concentration in a biological sample of a        peptide comprising the same or substantially the same amino acid        sequence as Seq ID no:1 or a fragment thereof;    -   (ii) comparing the peptide concentration determined in step (i)        with one or more reference values.

According to yet another aspect, the present invention provides a methodof determining the prognosis for a subject with osteoarthritis and/oranother ageing-related disease, comprising the steps of:

-   -   (i) determining the concentration in a biological sample of a        peptide comprising the same or substantially the same amino acid        sequence as Seq ID no:1 or a fragment thereof;    -   (ii) comparing the peptide levels determined in step (i) with        one or more reference values.

According to a further aspect, the present invention provides a methodof determining the efficacy of a treatment for osteoarthritis and/oranother ageing-related disease in a subject, comprising the steps of:

-   -   (i) determining the concentration in a biological sample of a        peptide comprising the same or substantially the same amino acid        sequence as Seq ID no:1 or a fragment thereof;    -   (ii) administering a treatment for osteoarthritis and/or another        ageing related disease to the subject;    -   (iii) determining after treatment, the concentration in another        biological sample of a peptide comprising the same or        substantially the same amino acid sequence as Seq ID no:1 or a        fragment thereof;    -   (iv) comparing the peptide concentrations determined in step (i)        and (iii) with one another, and optionally with one or more        reference values.

Preferably, in any method of the invention, the concentration of apeptide with the same sequence as the sequence of Seq ID no: 1 isdetermined. Alternatively, the concentration of a peptide substantiallythe same as the sequence of Seq ID no: 1 may be determined.

Alternatively, the concentration of a peptide fragment having a sequencethe same, or substantially the same, as part of the sequence of Seq IDno: 1 may be determined. Preferably if the concentration of a fragmenthaving a sequence the same, or substantially the same, as part of thesequence of Seq ID no: 1 is determined, the fragment represents anepitope within the sequence of Seq ID no: 1. Preferably the peptidefragment is at least 10, preferably at least 20, more preferably atleast 30 amino acids long.

Alternatively to a peptide or a peptide fragment one may also consider afree fragment which is intended to refer to a polypeptide, a peptide orotherwise released from mammalian serpine B1 molecule by an oxidative orenzymatic processing. A free fragment is different from a native proteinby its structure and configuration and may undergo modification such asphosphorylation, glycosylation or any other post-traductionalmodification resulting of a pathological mechanism. The free fragment aswell as the peptide or peptide fragment contributes to theidentification of the pathologic status of osteoarthritis patient.

An epitope is a binding site of an antibody on an antigen. In a peptideantigen, generally a linear epitope will be at least about 7 amino acidsin length, and may be at least 8, at least 9, at least 10, at least 11,at least 12, at least 14, at least 16, at least 18, at least 20, atleast 22, at least 24, or at least 30 amino acid residues in length.However, antibodies may also recognise conformational determinantsformed by non-contiguous residues on an antigen, and an epitope cantherefore require a larger fragment of the antigen to be present forbinding, e.g. a domain.

Reference herein to “a sequence substantially the same as” all or partof the sequence of Seq ID no: 1, refers to a peptide with a sequencewhich has at least 80%, preferably at least 90%, more preferably atleast 95% or 98%, sequence identity with all or part of the sequence ofSeq ID No:1. Preferably the sequence is the same or substantially thesame as at least about 10, 15, 20, 25, 30, 35, 40, 45 or moreconsecutive amino acids on the sequence of Seq ID No:1

Homology or sequence identity of two or more amino acid sequences can bemeasured by using a homology scoring algorithm NCBI BLAST (NationalCenter for Biotechnology Information Basic Local Alignment Search Tool).Alternatively, the UWGCC Package provides the BESTFIT program which canbe used to calculate sequence identity between two or more sequences(e.g. used on its default setting) (Devereux et al (1984) Nucleic AcidsResearch 12 p 387-395).

The sequence of Seq ID no:1 represents a fragment of the serpin B1protein. The fragment may be a degradation product of serpin B1.

In any method of the invention the peptide comprising a sequence thesame or substantially the same as the sequence of Seq ID no: 1 or a partthereof is preferably differentially present in the sample from asubject with osteoarthritis or another ageing-related disease comparedto a normal subject.

A peptide comprising the same or substantially the same sequence as SeqID no: 1 or a fragment thereof, which is measured in step (i) and/orstep (iii) in any method of the invention, is also referred to herein asthe biomarker or the biomarker peptide.

Reference to the “osteoarthritis status” or to the “status of anageing-related disease” refers to any distinguishable manifestation ofosteoarthritis or the ageing-related disease, including diseased andnon-diseased. For example, osteoarthritis status includes, withoutlimitation, the presence or absence of osteoarthritis in a subject, therisk of a subject developing osteoarthritis, the stage of the disease,the progression of the disease, and the effectiveness or response of asubject to treatment for osteoarthritis.

Any method of the invention may be used in conjunction with theassessment of clinical symptoms and/or imaging results and/or theconcentration of one or more other biomarkers.

Preferably all methods of the invention are carried out in vitro.

The biological sample may comprise urine, whole blood, blood serum,blood plasma, synovium, sweat, cerebrospinal fluid, mucous, saliva,lymph, bronchial aspirates, milk and the like. Preferably the biologicalsample is urine.

Biological sample, such as urine, have the advantage of being abundantand easily accessible.

A further advantage of using a biological sample, compared to a tissuesuch as cartilage, is that the progression of osteoarthritis or anotherageing-related disease, and/or the therapeutic effect of a treatment,may be monitored by taking and testing samples as often as necessarywithout the need for invasive procedures.

The concentration of the biomarker peptide in a sample may be determinedby any suitable assay. A suitable assay may include one or more of thefollowing methods, an enzyme assay, an immunoassay, mass spectrometry,HPLC, electrophoresis or an antibody microarray, or any combinationthereof. If an immunoassay is used it may be an enzyme linkedimmunoassay (ELISA), a sandwich assay, a competitive assay, aradioimmunoassay, a Western Blot, an immunoassay using a biosensor, animmunoprecipitation assay, an agglutination assay, a turbidity assay ora nephlelometric assay. If mass spectrometry is used it may be MatrixAssisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) MassSpectrometry.

Preferably the concentration of the biomarker peptide is determinedusing an immunoassay which uses one or more antibodies directed to thespecific biomarker peptide to determine the concentration of thebiomarker peptide in the sample.

If one or more antibodies are used to determine the concentration of abiomarker peptide in a sample the one or more antibodies may besynthetic, monoclonal, polyclonal, oligoclonal, bispecific, chimericand/or humanised.

One or more of the antibodies used may comprise a tag or a label. Thetag or label may be selected from the group comprising a radioactive, afluorescent, a chemiluminescent, a dye, an enzyme, or a histidine tag orlabel, or any other suitable label or tag known in the art.

Preferably the reference value, to which the determined concentration ofthe biomarker peptide is compared, is the concentration of the samepeptide in one or more “normal” subjects that do not have any detectableosteoarthritis and/or other ageing-related disease, or any clinicalsymptoms of osteoarthritis and/or other ageing-related disease, and haveso called “normal values” of the biomarker peptide.

Alternatively, the reference value may be a previous value for thebiomarker peptide obtained from a specific subject. This kind ofreference value may be used if the method is to be used to monitorprogression of osteoarthritis and/or another ageing-related disease, orto monitor the response of a subject to a particular treatment.

When the determined concentration of the biomarker is compared with areference value, an increase or a decrease in the concentration of thebiomarker may be indicative of the osteoarthritis status, and/or thestatus of another ageing-related disease, in the subject.

More specifically an increase or a decrease in the concentration of thebiomarker may be indicative, or diagnostic, of osteoarthritis in thesubject. Preferably, a decrease in the concentration of the biomarker,preferably of a peptide with the sequence of Seq ID no:1, in a sample isdiagnostic of osteoarthritis.

Preferably an at least about 5 fold or more increase in theconcentration of a peptide with the sequence of Seq ID no:1, or sequencesubstantially the same as Seq ID no:1, or a fragment thereof, in asample compared to a reference sample from a normal subject isdiagnostic of osteoarthritis. Preferably, an at least about 5.8 fold,decrease in the concentration of a peptide with the sequence of Seq IDno: 1, or sequence substantially the same as Seq ID no: 1, or a fragmentthereof, in a sample compared to a reference sample from a normalsubject is diagnostic of osteoarthritis.

The method of the invention may also be used to monitor osteoarthritisprogression, and/or the progression of another ageing-related disease,in a subject. Furthermore, the method of the invention may be used tomonitor the efficacy of a treatment for osteoarthritis, and/or anotherageing-related disease, following administration of the treatment to asubject. Efficacy of a treatment may be monitored by analysing samplestaken from a subject at various time points following initiation of thetreatment. By monitoring changes in the concentration of the biomarkerpeptide over time and comparing these concentrations to normal and/orreference values, efficacy of the treatment may be determined.

In this case reference concentrations may include the initialconcentration of the biomarker peptide in the subject, or theconcentration of the biomarker peptide in the subject when they werelast tested, or both.

According to another aspect of the invention there is provided a kit foruse in determining the osteoarthritis status, or the status of anotherageing-related disease, in a subject comprising at least one agent fordetermining the concentration of a peptide comprising the same orsubstantially the same amino acid sequence as the sequence of Seq ID No:1, or a part thereof, in a sample provided by the subject.

The kit may be used to diagnose osteoarthritis and/or anotherageing-related disease in a subject. The kit may alternatively be usedto monitor disease progression or the efficacy of a treatmentadministered to a subject with osteoarthritis and/or anotherageing-related disease.

The agent may be an enzyme, an antibody, a protein probe, a metaboliteor any other suitable composition.

The agent for determining the concentration of one or more biomarkerproteins is preferably labelled. The kit may also comprise means fordetecting the label.

The kit may comprise one or more capture agents for capturing thepeptide comprising the same or substantially the same amino acidsequence as the sequence of Seq ID No:1, or a part thereof, in a sampleprovided by the subject. The capture agent may be one or moreantibodies. The capture agent may be an antibody according to an aspectof the invention.

The kit may comprise two antibodies for use in a sandwich assay todetermine the concentration of a peptide comprising the same orsubstantially the same amino acid sequence as the sequence of Seq IDNo:1, or a part thereof. Preferably the kit comprises two antibodies,each directed to a different epitope on the peptide comprising the sameor substantially the same amino acid sequence as the sequence of Seq IDNo:1, or a part thereof. One antibody is preferably the captureantibody, and the other may comprise a label to allow its detection.

The capture agent may be attached to a solid support. The solid supportmay be a chip, a microtitre plate, a bead or a resin.

The kit may comprise instructions for suitable operational parameters inthe form of a label or separate insert. The instructions may inform auser about how to collect the sample, and/or how to wash the captureagent.

The kit may comprise samples of the biomarker peptide to be detected.The samples of the biomarker peptide may be used as a standard forcalibration and comparison. The kit may also comprise instructions tocompare the concentration of the biomarker peptide detected in a samplewith a calibration sample or chart. The kit may also includeinstructions indicating what concentration of the biomarker peptide isdiagnostic of osteoarthritis and/or another ageing-related disease.

According to a yet further aspect, the invention provides the use of thedetermination of the concentration of the biomarker peptide in abiological sample of as a means of assessing the osteoarthritis statusin a subject or as a means of assessing the status of anotherageing-related disease in a subject.

According to a yet further aspect, the present invention provides theuse of a biological sample, such as urine, as a source of at least onebiomarker for the prognosis of osteoarthritis progression and/or anotherageing-related disease, for diagnosing osteoarthritis and/or anotherageing-related disease and for monitoring the effect of a treatment forosteoarthritis and/or another ageing-related disease.

According to another aspect the invention provides a peptide, whichcomprises the same or substantially the same amino acid sequence as theamino acid sequence of Seq ID NO: 1, or a part thereof, or its amide, ora salt thereof.

Preferably the peptide of the invention has a sequence the same as thatof Seq ID No: 1.

According to a further aspect, the invention provides a polynucleotideencoding a peptide according to the invention. Preferably, thepolynucleotide is a DNA molecule.

According to a yet further aspect the invention provides a recombinantvector, which comprises a polynucleotide of the invention.

According to another aspect the invention provides transformant, whichis transformed with the recombinant vector of the invention.

In a further aspect, the invention provides the use of a peptideaccording to the invention in the manufacture of an antibody.

It a yet further aspect, the invention provides an antibody specific fora peptide according to the invention. In particular, the inventionprovides an antibody specific for a peptide having the sequence of SeqID No: 1.

An antibody according to the invention may be synthetic, monoclonal,polyclonal, oligoclonal, bispecific, chimeric or humanised. The antibodymay be complete or a fragment thereof, such as, Fv, Fab and F(ab)₂fragments. Methods of generating antibodies are well known in the art,and may include immunisation of suitable animals, such as, a rabbit,mouse, sheep or goat, with the peptide of interest (or an immunogenicfragment thereof) or recombinant techniques.

The skilled man will appreciate that preferred features of any oneembodiment and/or aspect of the invention may be applied to all otherembodiments and/or aspects of the invention.

Embodiments of the invention will now be described merely by way ofexample with reference to the accompanying figures in which:

FIG. 1A—shows an enlargement of a portion of a 2D-DIGE map fromosteoarthritis (OA) subject (right) and non-osteoarthritis (NO) subject(left).

FIG. 1B—shows a representation of spot 351 volume variation between OAsubjects (right) and non-osteoarthritis subjects (left).

FIG. 2—shows a graphic view of serpin B1 abundance modification based onthe spot volume decrease shown in FIG. 1B.

FIG. 3A—shows the result of a mass spectrometry analysis of trypticfragments of the peptide of serpin B1 recovered from spot 351 detailedin FIG. 1A, FIG. 1B and FIG. 2.

FIG. 3B—shows the sequence of human serpin B1 containing the fragmentsshown in FIG. 3A. (SEQ ID NO 3)

FIG. 4—shows the protein sequence of SEQ ID No. 1.

FIG. 5—shows the protein sequence of SEQ ID No. 2.

2D-DIGE (two dimensional difference gel electrophoresis—Marouga et al,(2005) Anal Bioanal Chem 382 (3): 669-78) methodology is a powerful toolfor investigating protein expression profiles in multiple sets ofsamples.

In this example, 2D-DIGE was used to study the protein expressionprofiles in urine samples from subjects with serious osteoarthritis andfrom healthy young subjects. Proteins in the samples to be compared werelabelled with either Cy3 or Cy5 CyDye DIGE Fluors. The Cy2 CyDye DIGEFluor was used to label a pooled sample comprising equal amounts of eachof the samples within the study, and this used as an internal standard.The use of this internal standard ensured that all proteins present inthe samples were represented, assisting both inter- and intra-gelmatching.

Materials and Methods Urine Samples Preparation

Urine samples were collected from 10 women undergoing hip replacementsurgery due to severe osteoarthritis. Control samples were obtained from5 healthy women (25.6±2.6 years) without articular degeneration. Theurine samples were concentrated 100× by ultracentrifugation on AmiconUltra-15 (Millipore, USA). Proteins were purified by precipitation usingPlusOne 2D Clean-up kit (GE Healthcare, Sweden). Albumin depletion fromurine samples was performed using affinity columns according to theMontage Albumin Deplete Kit (Millipore, USA) manual utilisation.

Labelling of Proteins with Cy3 and Cy5 Dyes

In all experiments, the purified proteins were labelled on lysineresidues with Cy3 or Cy5 CyDye DIGE Fluors. The samples were minimallabelled which means that the ratio of dye to protein used was such thateach protein molecule was labelled with only one dye molecule. Threegels were made as shown in Table 1. Proteins from different samples werelabelled with Cy3 or Cy5 and loaded on the same gel. On the first andsecond gels, proteins from NO (normal) samples were labelled with Cy3CyDye DIGE Fluor whereas proteins from osteoarthritis (OA) samples werelabelled with Cy5 CyDye DIGE Fluor. Conversely, proteins from NO sampleswere labelled with Cy5 CyDye DIGE Fluor and proteins from OA sampleswere labelled with Cy3 CyDye DIGE Fluor on the third gel. An internalstandard (MIX) comprising equal amounts of NO and OA samples waslabelled with Cy2 CyDye DIGE Fluor and loaded on each gel.

TABLE 1 Gel 1 Gel 2 Gel 3 Cy3 NO NO OA Cy5 OA OA NO Cy2 MIX MIX MIX

In Table 1 OA means samples from subject with osteoarthritis, and NOmeans control samples from normal subjects.

Two-Dimensional Electrophoresis

Protein samples (37.5 μg) labelled with Cy3, Cy5 or Cy2 DIGE Fluor wereseparated by 2D electrophoresis using an IEF (ioselectric focusing)buffer (8 M urea, 2% CHAPS, 0.5% immobilized pH gradient [IPG] buffer,1% DTT, and trace of bromophenol blue) which was loaded into animmobiline DryStrip (pH 3-10 NL, 24 cm) (GE Healthcare, Sweden). Thefirst dimension isoelectric focusing was performed for 70,000 Vhr usinga Protean IEF Cell (Biorad) at 20° C. Next, the gels were equilibratedfor 12 minutes in equilibration buffer I (375 mM Tris-Cl [pH 8.8], 6 Murea, 20% glycerol, 2% SDS, and 130 mM DTT) and II (375 mM Tris-Cl [pH8.8], 6 M urea, 20% glycerol, 2% SDS, and 135 mM IAA). The seconddimension was run according to the Ettan DALTsix Electrophoresis Unitoperating manual (GE Healthcare, Sweden). A 12.5% SDS-polyacrylamideslab gel (24 cm) was used for the second-dimension gel electrophoresis.The IPG strips were placed on the surface of the second-dimension gel.The gels were then placed in SDS electrophoresis buffer (25 mM Trisbase, 192 mM glycine, 0.1% SDS) and run overnight at 1.5 W per gel.

Gels were scanned while still between two low-fluorescence glass platesusing a Typhoon 9400 fluorescent scanner and saved in .gel format usingImageQuant software (GE Healthcare, Sweden). The excitation wavelengthsfor Cy3 and Cy5 are 550 nm and 645 nm, and the emission wavelengths are570 nm and 670 nm for Cy3 and Cy5, respectively. The excitation/emissionwavelength of Cy2 is around 489/505 nm. Image analysis was performed onDeCyder™ software (GE Healthcare, Sweden). Interesting spots withdifferential fluorescent intensity between Cy3 and Cy5 were picked outthe gel in order to allow protein identification after post-stainingwith Coomassie Blue.

DeCyder 2D v6.5 software (GE Healthcare, Sweden) was used forsimultaneous comparison of abundance changes across sample groups. TheDeCyder differential in-gel analysis (DIA) module generated ratios foreach protein “spot” by comparing Cy3 and Cy5 signals to the Cy2 controlsignal. The DeCyder biological variation analysis module matched allprotein spot maps from the gels and normalized the DIA-generated Cy3:Cy2and Cy5:Cy2 ratios relative to the Cy2 signals for each resolved featureseparately. This enabled the calculation of average abundance changesacross all three samples within each test group, and the application ofunivariate statistical analyses (Student's t-test, ANOVA).

Proteins Identification

Protein spots were cut out of the polyacrylamide gel and washed twicefor 5 minutes with an ammonium hydrogenocarbonate (50 mM)-acetonitrilemix (1:1). Gel spots were incubated in dithiothreitol (10 mM), NH₄HCO₃(50 mM), for 40 min in a 56° C. water bath. Proteins in the gel spotswere alkylated for 1 h in the dark with iodoacetamide (55 mM) in NH₄HCO₃(50 mM). The gel spots were then washed twice with an ammoniumhydrogenocarbonate (50 mM)-acetonitrile mix (1:1), dehydrated withacetonitrile, and then dried for 15 minutes at room temperature. The gelspots were then rehydrated for 10 minutes on ice with modified trypsin(10 ng/μl) in NH₄HCO₃ (25 mM) and then incubated overnight at 37° C.Hydrolysis of peptides was stopped in TFA (1%)-ACN (5%) solution. Thegel spots were then sonicated twice for 1 minute in order to releaseprotein fragments out of the isolated gel spots. Protein fragments insolution were freeze-dried. The identity of proteins was determined bytandem mass spectrometry (MS-MS spectrometry). The Mowse score (Pappinet al (1993) Curr Biol June 1; 3 (6):327-32) gave the fidelity ofidentification.

Results

Proteins isolated from the urine samples and labelled with Cy3 or Cy5were separated by two-dimensional electrophoresis. The first separationwas performed with an isoelectric focusing range of pH 3-10 NL and aload of 37.5 μg of protein. 372 spots of proteins were matched betweenall gels. Spots with a modification of intensity between OA and NO witha ratio superior to 1.5 (t-test: p<0.05) were selected for proteinidentification using mass spectrometry. Table 2 shows the results ofanalysis of these spots, and details the size of the spot, the Mowsescore (which is −10 log (P) where P is the probability that the observedmatch is a random event), the abundance ratio, the name of the proteinidentified in the spot and the accession number for the identifiedprotein in the Swiss Prot database.

TABLE 2 Abun- dance Sequence ratio Spot coverage Mowse (OA/ Swiss-Protn^(o) (%) score NO) Protein description Accession 40 9 390 1.83 Poly-Igreceptor P01833 (PIGR) 43 5 159 1.6 Poly-Ig receptor P01833 (PIGR) 75 10340 −1.68 Transferrin P02787 219 11 334 −1.7 Kininogen-1 precursorP01042 6 55 Alpha 1 anti-trypsin P01009 (A1AT) 222 15 349 −1.64Kininogen-1 precursor P01042 7 99 A1AT P01009 223 13 405 −1.89Kininogen-1 precursor P01042 16 311 A1AT P01009 226 18 334 −1.73 A1ATP01009 6 219 Kininogen-1 precursor P01042 262 13 368 −1.91 Kininogen-1precursor P01042 6 61 A1AT P01009 267 10 304 −1.98 Kininogen-1 precursorP01042 269 5 143 −1.83 Kininogen-1 precursor P01042 343 189 4.18Beta-actine 348 21 192 2 Zn-α-2-glycoprotein P25311 precursor 349 8 195−2.44 Serpin B3 P29508 351 6 115 −5.84 Serpin B1 P30740 352 5 130 2.2Fibulin-3 Q12805 73 Apoptosis-inducing Q9BRQ8 factor 2 (two proteinsidentified in the same spot) 356 8 110 2.01 Zn-α-2-glycoprotein P25311 245 precursor Q12805 FIBULIN3 386 10 197 1.54 GP36b Q12907 398 5 187 2.28AMBP protein P02760 precursor 485 8 289 −2.3 Mannan-binding lectinO00187 serine protease 2 E.C precursor 3.4.21.104

As can be seen from Table 2, various proteins with significant changesin concentration in samples from osteoarthritis compared to samples fromnormal subjects were identified. Some of the proteins identified areimplicated in the inflammatory process, for example, the kininogenprecursor or alpha-1-antitrypsin. This observation coincides with thepathology of osteoarthritis.

A significant decrease in the concentration of a specific serpin B1fragment was observed in osteoarthritis subjects compared to normalsubject, as shown in Table 2 and FIGS. 1A and 1B. In FIG. 1A there isshown an enlargement of the area around spot 351 on the 2D-DIGE map ofproteins extracted from urine from osteoarthritis subjects (right) and aNO subjects (left). In FIG. 1B the same spot (spot 351) is representedby volume variation of the spot between samples from OA subjects (right)and NO subjects (left). A graphic view of the abundance modificationbased on the spot 351 volume decrease is also shown in FIG. 2.

Tryptic fragments from spot 351 were identified by mass spectrometryanalysis as shown in FIG. 3A. These fragments were identified as beingfragments from the protein serpin B1, as shown in FIG. 3B. FIG. 3B showsthe protein sequence derived by the translation of the mRNA of humanserpin B1 and in bold the specific sequence identified by massspectrometry. Each tryptic fragment studied was given a score which is−10 log(P) where P is the probability that the observed match is arandom event. Individual ion scores >26 indicate identity or extensivehomology.

The serpins are a superfamily of serine protease inhibitors (SERineProtease Inhibitors). In human, serpin family is divided, into nineclades (A-I). These proteins are involved in a diverse set of processesincluding blood coagulation, inflammation and complement activation,cell migration, pro-hormone activation and matrix remodelling. Theyundergo a unique and dramatic conformational change when they inhibittarget proteases. Serpines inhibit protease with substrate-likeirreversible inhibitory mechanism. Not all serpins functions asproteinase inhibitors. Some of them act as chaperone proteins (heatshock protein 47) or are involved in blood pressure regulation(angiotensinogen).

Clade B represents a particular set of serpines, ov-serpines, with aminoacid similarities among chicken ovalbumin. Most serpins target serineprotease, however, some serpines from clade B can inhibit serine orcysteine protease or both.

The serpin B1 or LEI (leukocyte elastase inhibitor) is involved inregulating the degradation of the extracellular matrix through itsaction on neutrophil elastase. The elastase is also involved in thepathogenesis of inflammatory diseases such as rheumatoid arthritis andhas been proposed as biomarker of chronic joint diseases (Kleesiek etal., 1986; Momohara et al, 1997). In the lung, administration ofrecombinant serpin B1 has a protective role against proteases releasedby neutrophils (Rees et al. 1999, Hirche et al., 2004; Rubio et al.,2004; and Yasumaki al., 2006).

An increased amount of serpin in plasma may be associated with aparticular pathology. For example, serpin B3 is associated with squamouscell carcinoma and serves as a diagnostic marker for some cancers(Pemberton et al., 1997).

1. A method of determining the osteoarthritis status of a subject,and/or the status of another ageing-related disease in a subject,comprising the steps of: (i) determining the concentration in abiological sample of a peptide comprising the same or substantially thesame amino acid sequence as Seq ID no:1 or a fragment thereof; (ii)comparing the peptide concentration determined in step (i) with one ormore reference values.
 2. A method of diagnosing osteoarthritis and/oranother ageing-related disease in a subject, comprising the steps of:(i) determining the concentration in a biological sample of a peptidecomprising the same or substantially the same amino acid sequence as SeqID no:1 or a fragment thereof; (ii) comparing the peptide concentrationdetermined in step (i) with one or more reference values.
 3. A method ofdetermining the prognosis for a subject with osteoarthritis and/oranother ageing-related disease, comprising the steps of: (i) determiningthe concentration in a biological sample of a peptide comprising thesame or substantially the same amino acid sequence as Seq ID no:1 or afragment thereof; (ii) comparing the peptide concentration determined instep (i) with one or more reference values.
 4. A method of determiningthe efficacy of a treatment for osteoarthritis and/or anotherageing-related disease in a subject, comprising the steps of: (i)determining the concentration in a biological sample of a peptidecomprising the same or substantially the same amino acid sequence as SeqID no:1 or a fragment thereof; (ii) administering a treatment forosteoarthritis and/or another ageing related disease to the subject;(iii) determining after treatment the concentration in anotherbiological sample of a peptide comprising the same or substantially thesame amino acid sequence as Seq ID no:1 or a fragment thereof; (iv)comparing the peptide concentrations determined in step (i) and (iii)with one another, and optionally with one or more reference values. 5.The method of claim 1 wherein the biological sample is selected from thegroup comprising urine, whole blood, blood serum, blood plasma,synovium, sweat, cerebrospinal fluid, mucous, saliva, lymph, bronchialaspirates and milk.
 6. The method of claim 1 wherein the concentrationof the peptide is determined by using an immunoassay.
 7. The method ofclaim 1 wherein the reference value is the concentration of the peptidein biological samples of one or more normal subjects.
 8. A kit for usein determining the osteoarthritis status, or the status of anotherageing-related disease, in a subject comprising at least one agent fordetermining the concentration of a peptide comprising the same orsubstantially the same amino acid sequence as the sequence of Seq IDNo:1, or a part thereof, in a biological sample.
 9. The kit according toclaim 8 wherein the agent is selected from the group comprising anenzyme, an antibody, a protein probe, a metabolite or any other suitablecomposition.
 10. The kit according to claim 8 wherein the agentcomprises two antibodies directed to different epitopes on the peptide.11. The kit according to claim 10 for use in determining theconcentration of the peptide by using a sandwich immunoassay.
 12. Anisolated peptide which comprises the same or substantially the sameamino acid sequence as the amino acid sequence of Seq ID NO: 1, or apart thereof, or its amide, or a salt thereof.
 13. The polynucleotideencoding a peptide according to claim
 12. 14. The recombinant vectorcomprising a polynucleotide according to claim
 13. 15. The transformanttransformed with a recombinant vector according to claim
 14. 16. The ofa peptide according to claim 12 in the manufacture of an antibody. 17.The antibody specific for a peptide according to claim 12.